DE2536713A1 - METHOD FOR DETERMINATION OF VITAMIN B-12 WITH RADIO ISOTOPES - Google Patents

Description

Method for the determination of vitamin B-12 with Eadioisotopes

The invention relates to a method for determining vitamin B-12 in a serum sample solution with radioactive isotopes, and in particular a method employing the principle of the eadioisotope dilution method in which improved operations for the separation of vitamin B-12 not bound by intrinsic factors (EF) as well as new formulations of reagent solutions and their order of addition can be applied to a faster and simpler process than has previously been the case analogous investigation procedures were used to obtain.

The principles of the process which the present invention is directed to improve are as follows Publications: K. S. Lau, C.W. Gottlieb, L. E. Wassermann and V. Herbert, "Measurement of Serum Vitamin B-12 Level Using Eadioisotope Dilution and Coated Charcoal ", Blood, 26 (1956), p. 202;

V. Herbert, C. W. Gottlieb and K. S. Lau "Hemoglobin-coated Charcoal Assay for Serum Vitamin B-12", Blood 28, (1966), p. 130;

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U.S. Patent 3,444-2,819.

In particular, that is used in the method according to the invention and principle known as radioisotope dilution in Example 9 of the aforementioned US Pat. No. 3,442,819 described. In this way of working, serum vitamin B-12 occurs and radioactive vitamin B-12 for the limited ability of self-factor (EF) to bind in competition. The amount the unbound, radioactive vitamin B-12 and unbound serum vitamin B-12 must be removed. A number of the previously indicated methods for removing or separating the bound vitamin B-12 from the free vitamin B-12 are given in the following publications:

Dialysis:

R. M. Barakat, and E. P. Ekins (1963) "An Isotopic Method for the Determination of Vitamin B-12 Levels in Blood ", Blood 21., pp.70-79;

S. Friedner, B. Josephson, and K. Levin (1969) "Vitamin B-12 Determination by Means of Radioisotope Dilution and Ultrafiltration » , Clin. Chim. Ac ta .24, pp. 171-179;

Diethylaminomethylcellulose (PEAΕ cellulose):

E. P. Urenkel, S. Keller, and M.S. McCaIl (1966) "Radioisotopic Assay of Serum Bitamin B-12 with the Use of DEAE-Cellulose ", J. Lab. Clin. Med. 68 pp. 510-522;

G.! Dibbling (1969) "A Method & r Determination of Vitamin B-12 in Serum by Radioassay ", Clin. Chim. Acta 2g, pp. 209-218;

Activated carbons coated with protein:

KS Lau, CV. Gottlieb, LR Vasserman and V. Herbert (1965), "Measurement of Vitamin B-12 Level Using Radioisotope Dilution and Charcoal ' ¹ , Blood 26, pp. 202-214;

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V. Herbert, C. W. Gottlieb, and K. S. Lau (1966), "Hemoglobincoated Charcoal Assay for Serum Vitamin B-12 ", Blood 28, pp.130-132;

J. L. Raven, P. L. Walker and P. Barkham (1966) "Comparison of the Eadioisotope Dilution-coated Charcoal Method and a Microbiological Method (L. leichmanni) for Measuring Vitamin B-12 in Serum ", J. Clin. Path. 1 £, pp. 610-613;

V. Herbert, U.S. Patent 3,442,819 (1969).

Precipitation of bound vitamin B-12;

S. P. Eothenberg (1963), "Eadioassay of Serum Vitamin B-12 by

57 Quantitating the Competition Between Co ^ '-B-12 and Unlabelled B-12 for the Binding Sites of Intrinsic Factor ", J. Clin. Invest. 42, pp. 1391-1398;

Sephadex;

L. Wide and A. Killander (1971), "A Eadiosorbent Technique for the Assay of Serum Vitamin B-12 ", Scand. J. Clin. Lab. Invest. 22, pp. 151-159

Eigenfactor linked to a solid phase (intrinsic factor); M. Cesca and U. Lundkvist, "Use of Solid Phase Intrinsic Factor for Eadiosorbent Assay of Vitamin B-12," Clin. Chim. Acta 32 , (197D, pp. 339-354;

Ion exchange resin:

PJ Brombacher, AHJ Gijzen, and MPJ Soons, "A Systematic Investigation on the Assay of Vitamin B-12 in Serum by Eadioisotope Dilution," Clin, Chim. Acta $ 6 (1972) pp 493-498;

P. Eoos in symposium on "In Vitro" working methods with Eadioisotopes in clinical medicine, Vienna 1969, IAEA-SM-124/27.

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All of these previously known ways of working for separation have certain compartment parts. For example, the use of activated charcoal solutions has detrimental effects, as is indicated in the article "The Separation of Free and Bound Vitamin B-12" in the British Journal of Hematology by JF Adams and C. McEwan Fiona 26 (197 ⁷ O, p.581 In addition, the main disadvantages of activated charcoal suspensions are the non-homogeneity of the dose and the introduction of additional, suspension-carrying volumes of fluid into each test tube. Column operations also require time-consuming elution and can generate large amounts of radioactive waste. Gel elution columns require an accurately reproducible set-up systems with solid phases such as immobilized eigenfactor (as are commercially available from pharmaceutical companies) require prolonged agitation (about three hours) of a solid / liquid system and subsequent washing of the solid support to remove during the Ze Centrifuge trapped material.

The object of the invention is to create a method that avoids the aforementioned difficulties.

Surprisingly, it has now been found that when using bentonite, preferably in the easily handled form a pre-made tablet, for the adsorption of unbound, radioactive vitamin B-12 and serum vitamin B-12 this Can avoid disadvantages. This used in the present invention Adsorbent is easily separated from the test medium by centrifugation and gives consistently high amounts of recovered material.

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According to the invention, therefore, serum vitamins B-12 and radioactive vitamin B-12 lead substance "or trace substance for the Eigenfactor (EF) with "limited binding capacity, i.e. with a lower binding capacity than it for Binding of total serum vitamin B-12 and total radioactive vitamin B-12 is required in competition. Thereafter, free, radioactive vitamin B-12 and free serum vitamin B-12 are bound by the intrinsic factor Vitamin B-12 before measuring radioactivity to match this with a obtained using vitamin B-12 standards To relate the curve, disconnected. The separation of free vitamin B-12 and free serum vitamin B-12 from that radioactive vitamin B-12 and the serum vitamin B-12, which are bound, is obtained by adding an effective amount of Bentonite is used to adsorb free, radioactive vitamin B-12 and free serum vitamin B-12, making the sample solution of the intrinsic factor (EF) with bound, radioactive vitamin B-12 and serum vitamin B-12 physically of solid Bentonite can be secreted with adsorbed free vitamin. This way the radioactivity is in the liquid Phase proportional to the amount of bound vitamin. The amount of bentonite added per test tube is not critical provided that every test tube is the same So that there is an advantage in tableting this mineral. One 10 mg tablet (not included inert carrier cellulose) is suitable for the sample size used.

Other new and advantageous aspects of the invention, improved test methods relate to the pre-combination of acidic test media and the radioactive lead substance or trace substance. These and others

- ο -

Combined Eeagentien, which are described in the following, result in a convenient implementation option for the personnel carrying out the investigation. In the case of this combination of investigation media and lead substance or trace substance the advantageous result is to be seen in the fact that the radioactive lead substance or trace substance is the same Treatment as the serum vitamin B-12 experiences. Other reagent solutions used include: HSA, pre-combined with standard, HSA pre-combined with EF and a cyanide reagent. The acidic medium in conjunction with the cyanide reagent and the heating step cause the release of vitamin B-12 and denaturation of accompanying binders. The latter reagents are used in terms of component concentrations formulated or scheduled so that an equal volume of each of the reagent solutions is used in the procedure the investigation is possible. This means that all reagent solutions which are different from the combined acidic investigation media and the lead substance or trace substance solution can be measured with a pipette of the same size. The number of liquid measurements is used for the Personnel reduced and extremely simplified.

1 shows a standard curve for use in the test method according to the invention. The values, which are obtained with / known serum can be applied to the Curve can be plotted to find the concentration of serum vitamin B-12.

In the following preferred embodiments of the invention Method explained in more detail using examples.

example 1

This example shows the implementation of the investigation methods with radioactive material. The following reagents were used:

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Reagent 1 is a tablet containing 10 mg of bentonite, inert binder and cellulose.

The reagent 2 is an intrinsic factor, which is set at 20 mg / 100 ml H ₂ O and filtered, and to which human serum albumin (HSA) was added ⁺ (.70 to .01 g HSA per 100 ml of sterile water). It is set so that a B of 50% (approximately) is achieved. Greater dilution may be required if B> 50 % . For this, reference is made to the calculation given _{below for the definition of B Q} under number 9.

The reagent 3 ⁺⁺ consists of 20% 0.25-0.02N HCl and 80%

approx

0.9% NaCl in water. Vitamin B-12-Co ^ 'becomes this Medium added before use and adjusted to be 65-3 pg / ml.

Reagent 4 is potassium cyanide (0.05 mg / ml) and it will be like prepared as follows: 100-105 mg of potassium cyanide are dissolved in sterile water, then 2.0 ml of an O, 1N Sodium hydroxide solution (4 g NaOH / 1) added and adjusted to 1000.0 i 0.1 ml made up with sterile water. For working dilution, dilute 1: 1.

+ * The HSA solutions contain 0.001 % thimerosal as an enhancement agent.

++ * Other media that can be used alternatively are:

(i} 1M acetic acid
ii) i
)

(g
(ii) 0.5M acetic acid
(iii) 0.25M glycine buffer, pH - 3.4

In general, regardless of the acid or buffer selected, it is used in an amount to achieve a pH of about 1.9-3 _? 5 to generate in the media after the reaction between the test medium, the serum and the intrinsic factor. The results obtained using acetic acid and glycine buffer as the releasing agent for vitamin B-12 are similar to those obtained using HCl / saline.

The investigation stages can be described as follows, mixing after each addition:

1. A number of test tubes are labeled 1 through N. numbered, and 1 ml of reagent 3 is added to each test tube.

2. Add 200 μl of the standard to test tubes 1 and 2 A and to the test tubes 3 to 7 200 ial of the standard solutions B to F added.

(a) Standard A contains 0 pg vitamin B-12 / ml

(b) Standards B to F have the following concentrations: B «100 pg B-12 / ml; C »250 pg B-12 / ml D «500 pg B-12 / ml; E - 1000 pg B-12 / ml F - 2000 pg B-12 / ml

3. Add 200 µl to test tubes 8 through H and following of the unknown sera were added.

4. To all test tubes add 200 µl of the reagent four times.

5. All test tubes are used in a water bath of 100 ^° C. for 15 ″ minutes.

6. The test tubes are removed and brought to room temperature cooled, 200 nl of reagent 2 are added to test tubes 2 to N and the following. It will JO minutes at ambient temperature (room temperature) ditched. No reagent 2 is added to the test tube 1.

7. At the end of the 30 minutes, an adsorbent tablet is added (Reagent 1) added to each test tube. Each test tube is shaken for 10 seconds and left to stand for 5 minutes and 10 minutes or a sufficient amount of time at one

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sufficient number of revolutions, ζ. Β. 2500, centrifuged to thoroughly settle the adsorbent.

8. The supernatant liquid is decanted into counting glasses and measured on a suitable instrument.

9. The calculations were carried out as follows: The ratio B / B is calculated.

B «Amount of radioactivity in the supernatant liquid of the serum or the standard test tube minus the radioactivity in the supernatant liquid of the Test tube with the blank sample;

B «Amount of radioactivity in the supernatant liquid of the test tube with the zero standard minus the radioactivity in the supernatant liquid of the test tube with the blank sample;

B / B ■ (percent of bound lead substance or trace substance in% of the zero standard) »

Ipm of test tube No. N _ Ipm of reagent or standard) "Glass No. 1 ^ ₀₀ pm of test tube No. 2 - Ipm of reagent

glass No. 1

The standard curve was obtained as follows: There are the values of B / B compared with the pg / ml value of Standards A through F applied, ζ. B. on double logarithmic paper.

The unknown values were obtained as follows:

There were pg / ml values from the standard curve using the B / B values, as obtained above read.

The addition of the reagents is summarized below:

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Reagent lead substance standard or KCN CuI) Eigenfactor glass No. (ml) Serum (^ a.1) ' CuI)

1, blank 1 200 200 sample

2, lead substance
bond 1 200 200 200

3-7, standard 1 200 200 200

8-N, sera 1 200 200 200

Let simmer for 15 minutes

Using the procedure outlined above, the data shown in Table I below were obtained. The default values were used to plot the curve shown in Figure 1 of the drawing:

Table I calculations

Reagent sample Ipm Ipm bound vitamin

glasses blind as% of B-12 in

worth Leiteub- picogram /

stanzbin ml from the

dunft (0 hrs.) curve

1A
1B Blank sample
glass jars 91)
110
128) 5665)
) 5640
5615) 100 % 2A
2 B Lead substance
dung (Kttllstaa—
dard) 5725 4310 76.4 % 3A 100 picograms /
ml standard 4420 4396 77.9% 3B 4506 3111 55.2 % 4A 250 picograrai /
ml standard 3221

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Test tubes

sample

Ipm Ipm- bound vitamin blind- as % of the B-12 in value Lei t sub- picograms / stanzbin- ml from the

dung (o hrs.) curve

4B 5A

5B 6A

6B 7A

7B 8A

8B 9A

9B

500 picograms / ml standard

1000 picograms / ml standard

2000 picograms / ml standard

unknown serum no.

unknown serum no.

3292 3182 56.4 2314 2204 39.1 2343 2233 39.6 1454 1344 23.8 1464 1354 24.0 816 706 12.5 887 in 13.8 1800 1690 30.0 1812 1702 30.2 2697 2587 45.9 2569 2459 43.6

740 730

385 420

Example 2

This example shows the superiority of bentonite as

Absorbent.

To add to this superior performance of bentonite in removing free vitamin B-12 from the reaction medium show, the following experiment was carried out:

To 1.0 ml of a solution with 0.20 ml of 0.255N HCl, 0.80 ml of 0.9%

NaCl solution and 65 pg vitamin B-12-Co ^ (100 nCi / ng) were 0.20 ml collected serum added. This was followed by the addition of 0.20 ml of a KCN solution (10 11 g of KCN) and heating for

Minutes to 100 ^° C.

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After cooling to room temperature, 0.010 g of a silicate mineral was added in each case and the test tubes were shaken for 10 seconds. The supernatant liquid was 1 minute after centrifugation at 2900 rpm for Counted 10 minutes.

The positive controls were carried out with

(i) Tetra Count Tablets ⁺
(ii) Activated charcoal, 10 mg / O, 8 ml of 0.9% NaCl solution

Table II

Mineral% of total radioactivity recovered

Dickit 3.1

Pyrophyllite 2.7

Metabentonite (potash bentonite) 13.8

Kaolinite 7.2

Attapulgite 24.3

Florisil 38.1

Bentonite, 99.6

Controls

Tetra Count tablets 99.2

Activated carbon suspension 98.0

+ Tetra-Count tablets are commercially available tablets from Bio-Rad Laboratories, Richmond, Kalif., V.St.A., which contain 10 mg bentonite.

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In the method according to the invention for radioactive examination on vitamin B-12, therefore, the unknown amount of non-radioactive vitamin B-12 is used, which from Serum mixed with the radioactivity of a "known amount of radioactive vitamin B-12 lead substance or trace substance is released. A solution of the intrinsic factor with a binding capacity less than the amount of the serum vitamin B-12 and the radioactive vitamin B-12, is used to bind part of the vitamin B-12 mixture. That Vitamin B-12 that is not bound to the eigen factor is removed by adding a tablet containing bentonite. the The amount of radioactive vitamin B-12 bound to the inherent factor is compared with, and based on, standard values In this way, the unknown values of the serum vitamin B-12 are obtained.

During the stages of the process, the acidic test medium is mixed with the radioactive lead substance or trace substance pre-combined or previously combined so that the radioactive vitamin B-12 lead substance or trace substance is the same Treatment as the serum vitamin B-12 experiences. Certainly the other reagent solutions are also pre-combined or previously combined, and the concentration of the constituents is so set the volume of each of these other reagent solutions used to be the same at the various stages of the assay is. In this way, fewer pipetting steps are required.

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Claims (1)

Claims 1./Method for the radioactive determination of vitamin B-12 in a serum sample solution in which a radioactive vitamin B-12 lead substance or trace substance and serum vitamin B-12 enter into competition for the eigenfactor (EP) with limited binding capacity, and then free, radioactive Vitamin B-12 and free serum vitamin B-12 from the intrinsic factor and the radioactive vitamin B-12 bound to it and the serum vitamin B-12 bound thereto prior to measuring radioactivity for correlation with standard values are separated, characterized in that free, radioactive vitamin B-12 and free serum vitamin B-12 from the inherent factor and the radioactive vitamin B-12 bound to it and the serum vitamin bound to it B-12 by adding an effective amount of bentonite to adsorb free, radioactive vitamin B-12 and free Serum vitamin B-12 is separated, whereby the supernatant solution of the inherent factor with the bound, radioactive Vitamin B-12 and the bound serum vitamin B-12 from the solid bentonite with adsorbed free vitamin B-12 and adsorbed free serum vitamin B-12 can be physically secreted. 2. A method for the separation of free vitamin B-12 and a blood serum solution, characterized in that an effective Amount of bentonite combined with this solution to adsorb vitamin B-12 not bound by serum protein and that the bentonite with this adsorbed vitamin B-12 is separated from the solution. 609611/0635 J. Method according to. Claim 2, characterized in that the Bentonite is combined by dispersing it with the blood serum solution, and that then the solution to bring about the related separation is centrifuged. 4. Improved radio testing method for vitamin B-12, characterized in that it comprises the following steps: preparing a first solution, which acid and a preselected one Contains amount of radioactive vitamin B-12, preparing a blood serum sample, combining a second solution, which contains cyanidienes, with the first solution and the Blood serum sample, heating of the last-mentioned combination of solutions to reduce the binding capacity of the Serum proteins for free vitamin B-12 from this, preparation of a third solution, which is natural and human Contains serum albumin, combining this third solution with a refrigerated combination of the first solution, the second Solution and the serum sample following the heating stage in order to obtain a proportion of the radioactive Binding vitamin B-12 and serum vitamin B-12 through the intrinsic factor, and separating the free, radioactive vitamin B-12 and the free serum vitamin B-12 from the radioactive vitamin B-12 bound by the intrinsic factor and the serum vitamin B-12 bound therefrom. 5. The method according to claim 4, characterized in that the concentration of the components of the blood serum sample, the the second solution and the third solution are adjusted so that that the volume used of each solution is the same. 6. The method according to claim 4, characterized in that the separation of free, radioactive vitamin B-12 and free serum vitamin B-12 from the bound radioactive vitamin B-12 and bound serum vitamin B-12 609811/0635 Treating the combination of the first, second and third solutions and the serum sample with an effective amount of bentonite for the adsorption of free, radioactive vitamins B-12 and free serum vitamin B-12 is performed. 7 · The method according to claim 4, characterized in that the first solution contains an acid in the form of hydrochloric acid, acetic acid or a glycine buffer, in which the acid content is so it is set that after the reaction between the first solution, the serum sample and the inherent factor, the pH value is about 1.9 to 5.5. 809811/0635